Derivation of human microglia from pluripotent stem cells

ABSTRACT

The present invention relates to methods for deriving human hematopoietic progenitors, primitive macrophages, and microglial cells from human pluripotent stem cells. In particular, provided herein are highly efficient and reproducible methods of obtaining human primitive macrophages and microglia from human pluripotent stem cells, where the primitive macrophages and microglia can be suitable for clinically relevant therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Ser. No.62/098,824, filed Dec. 31, 2014, which is incorporated herein as if setforth in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

This invention was made with government support under TR000506 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Pluripotent stem cells offer a potentially powerful tool for improvingin vitro models and investigating the underlying mechanisms ofdevelopment of human neural tissue and of neurotoxicity. Animal modelshave provided insight into these mechanisms, but are of limited valuefor predicting developmental neurotoxicity due to poorly understooddifferences in the human brain such as an expanded cerebral cortex.There exists substantial academic literature supporting the theory thatmicroglia arise from the transition of a primitive myeloid progenitorcell population during early development. As the neural tube begins toform, it has been shown in rodent models that early yolk sachematopoietic progenitors begin to migrate to the anterior position ofthe neural tube and form resident populations of immune-like cells thatlater develop into microglia. The majority of studies have beenperformed in rodent models, leaving much to be desired for the study ofthe human cell type. Accordingly, there remains a need in the art, forefficient, reproducible, and xenogeneic material-free methods fordifferentiating human pluripotent stem cells into microglia suitable forclinical cell therapies and for predictive analysis of candidateneurotoxic agents.

SUMMARY OF THE INVENTION

In a first aspect, this document provides a method of obtaining humanhematopoietic precursor cells. The method generally comprises culturinghuman pluripotent stem cells under normoxic conditions for about 24hours, where the pluripotent stem cells are cultured on a substrate thatpromotes cell adhesion and in a culture medium consisting essentially ofL-ascorbic acid-2-phosphate magnesium, sodium selenium, transferrin,insulin, NaHCO₃, fibroblast growth factor 2 (FGF2), transforming growthfactor beta 1 (TGFβ1), and a Rho kinase (ROCK) inhibitor, whereby thecultured pluripotent stem cells differentiate into hematopoieticprecursor cells (HPCs). The substrate that promotes cell adhesion cancomprise Tenascin-C. The Tenascin-C can be recombinant human Tenascin-C.The ROCK inhibitor can be selected from the group consisting of Y-27632,Blebbistatin, HA-1077.

In another aspect, this document provides a method of obtaining humanmyeloid progenitors. The method generally comprises culturing human HPCsobtained according to a method provided herein for about 3 to about 5days in a culture medium comprising FGF2, a vascular endothelium growthfactor (VEGF), thrombopoietin (TPO), stem cell factor (SCF),interleukin-6 (IL-6), and interleukin-3 (IL-3), where the hematopoieticprogenitor cells differentiate into myeloid progenitors.

In yet another aspect, provided herein is a method of obtaining humanprimitive macrophages. The method generally comprises culturing humanmyeloid progenitors obtained according to a method provided herein inthe presence of a culture medium comprising insulin and a hematopoieticcytokine, whereby the cultured myeloid progenitors differentiate into acell population comprising at least 80% CD45⁺/CD11b⁺/CD14⁺ primitivemacrophages. The CD45⁺/CD11b⁺/CD14⁺ primitive macrophages can beCD34^(low/negative). In some cases, the CD45⁺/CD11b⁺/CD14⁺ primitivemacrophages do not express a detectable level of Iba-1. Thehematopoietic cytokine can be granulocyte macrophage colony-stimulatingfactor (GM-CSF).

In another aspect, this document provides a method of obtaining humanhematopoietic precursor cells. Generally, the method comprises the stepsof (a) culturing human pluripotent stem cells under hypoxic conditionson a substrate that promotes cell adhesion and in a growth mediumconsisting essentially of Dulbecco's Modified Eagle Medium (DMEM),nutrient mixture F12, a chemically defined lipid concentrate, L-ascorbicacid-2-phosphate magnesium, monothioglycerol, sodium selenium, polyvinylalcohol, L-alanyl-L-glutamine, FGF2, bone morphogenetic protein 4(BMP4), Activin A, and an inhibitor of glycogen synthase 3 (GSK3) for alength of time between about 40 and about 48 hours, where thepluripotent stem cells are initially seeded on the substrate at a celldensity between about 2×10⁵ cells per cm² and about 2.5×10⁵ cells percm²; and (b) further culturing the cultured cells of step (a) underhypoxic conditions in a culture medium comprising FGF2, a VEGF, and aninhibitor of TGFβ-mediated signaling, whereby the further cultured cellsdifferentiate into hematopoietic progenitor cells. The substrate thatpromotes cell adhesion can comprise vitronectin. The inhibitor of GSK3can be selected from the group consisting of CHIR99021, lithium chloride(LiCl), and 6-bromoindirubin-3′-oxime (BIO). The inhibitor ofTGFβ-mediated signaling can be selected from the group consisting ofSB431542 and A-83-01.

In a further aspect, provided herein is a method of obtaining humanmyeloid progenitors. The method generally comprises culturing human HPCsobtained according to a method provided herein under normoxic conditionsin a culture medium comprising FGF2, a VEGF, TPO, SCF, IL-6, and IL-3until cultured hematopoietic progenitor cells differentiate into myeloidprogenitors.

In another aspect, this document provides a method of obtaining humanprimitive macrophages. The method generally comprises culturing humanmyeloid progenitors obtained according to a method provided herein undernormoxic conditions in the presence of a culture medium comprisinginsulin and a hematopoietic cytokine, whereby the cultured myeloidprogenitors differentiate into a cell population comprising at least 80%CD45⁺/CD11b⁺/CD14⁺ primitive macrophages. The hematopoietic cytokine canbe human granulocyte macrophage colony-stimulating factor (GM-CSF). TheCD45⁺/CD11b⁺/CD14⁺ primitive macrophages can be CD34^(low/negative). Insome cases, the CD45⁺/CD11b⁺/CD14⁺ primitive macrophages do not expressa detectable level of Iba-1.

In another aspect, this document provides a method of making acomposition comprising human microglial cells, the method comprisingcontacting human pluripotent stem cell-derived primitive macrophages toa chemically defined, xenogen-free three-dimensional tissue constructcomprising stratified layers of human neurons and glia, therebyproducing a composition comprising human microglial cells. The primitivemacrophages can be obtained according to a method described herein. Thetissue construct can comprise a hydrogel. The microglial cells can beIba-1⁺. Prior to the contacting step, the human primitive macrophagescan be cultured for about 5 days in a culture medium consistingessentially of DMEM/F12, interleukin-1-beta (IL-1β), serum, and ahematopoietic growth factor. The hematopoietic growth factor can bemacrophage colony-stimulating factor (M-CSF).

In yet another aspect, this document provides a method of screening acompound for toxicity. Generally, the method comprises exposing a testcompound to a composition obtained according to a method provided hereinand assaying for an effect of the compound on one or more aspects ofhuman microglial growth or development.

These and other features, objects, and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

This application includes a sequence listing in computer readable form(a “txt” file) that is submitted herewith. This sequence listing isincorporated by reference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIGS. 1A-1B depict neural construct formation on synthetic poly(ethyleneglycol) hydrogels. (A) Hydrogels were formed using “thiol-ene”photopolymerization to crosslink 8-arm poly(ethylene glycol)-norbornene(PEG-NB) molecules with cysteine-flanked matrix metalloproteinase(MMP)-degradable peptides, while pendant CRGDS peptides wereincorporated for adhesion. (B) Human embryonic stem cell-derivedprecursor cells were cocultured on PEG hydrogels in 24-well transwellinserts. Neural progenitor cells (NPCs) were seeded on synthetic PEGhydrogels (Day 0), followed by endothelial cells (ECs) and mesenchymalstem cells (MSCs) at Day 9 and microglia/macrophage precursors (MG) atday 13.

FIGS. 2A-2G depict capillary networks and microglia withinthree-dimensional neural tissue constructs. Immunofluorescence wasdetected using antibodies specific for endothelial cells (CD31, green),glial cells (GFAP, red), and nuclei (DAPI, blue). Maximum projectionz-stacks for neural constructs at (A) day 1 and (B-G) day 21. Magnifiedportions of (B) are shown in (C) and (D) as labeled. Magnified regionsof (C) are shown in (E) and (F) as labeled. All images: Arrows showassociations between CD31⁺ endothelial cells and GFAP⁺ glial cells.Scale bars: (A-C) 100 μm; (D) 50×50 μm; (E-F) 25×50 μm; (G) 50 μm.

FIGS. 3A-3D depict microglia marker expression patterns, ramifiedmorphology, and capillary tubule interactions of microglia/macrophageprecursors. (A) Expression of microglia markers within neural constructsat days 16 and 21. Values shown are average transcripts per million(TPM) for control constructs from toxicity experiment ±S.D. (N=4). (B,C) Immunofluorescence images illustrating microglia (Iba1, red),endothelial cells (CD31, green), glial cells (GFAP, white), and nuclei(DAPI, blue): (B) Microglia (Iba1, red) with ramified morphologies; (C)Microglia (Iba1, red) interacting with endothelial cells (CD31, green);(D) Microglia (red), endothelial cells (green), and glial cells (white)at the leading edge of an extending capillary tubule. Inset illustratesa projection image for fewer slices and without GFAP to highlightinteractions of Iba+ microglia with the extending tubule. Scale bars: 50μm.

FIGS. 4A-4B present flow cytometry data from human pluripotent stemcell-derived primitive macrophages (day 6). Quadrants depict (A) CD14and CD11b expression and (B) CD45 and CD11b expression.

FIGS. 5A-5B present flow cytometry data from hematopoietic precursorcells (HPCs) and myeloid precursors. Quadrants depict (A) CD14 and CD34expression and (B) CD45 and CD34 expression.

FIGS. 6A-6D present flow cytometry data characterizing human pluripotentstem cell-derived primitive macrophages. Quadrants depict (A) CD68 andCD11b expression; (B) CD14 and CD11b expression; (C) CD45 and CD11b; and(D) NG2 and CD11b.

FIGS. 7A-7J present images demonstrating that three-dimensional (3D)neuronal constructs form interconnected vasculature and induce microgliadifferentiation. Maximum intensity Z-projection immunofluorescenceimages illustrating vascular networks and microglia within neuronalconstructs at (A-C) Day 16 and (D-H) Day 21. Vascularization at day 16for (A) a full neuronal tissue construct illustrating endothelial cells(CD31, green) and nuclei (DAPI, blue) and (B, C) zoomed imagesillustrating endothelial cells (CD31, green), glial cells (GFAP, red)within the boxed regions shown in (A). Vascularization at day 21 for (D)a full neuronal tissue construct illustrating endothelial cells (CD31,green) and nuclei (DAPI, blue) and (E, F) zoomed images illustratingendothelial cells (CD31, green), glial cells (GFAP, red) within theboxed regions shown in (D). (G) A reconstructed z-stack (boxed region inF) illustrating capillary tubule formation in distinct layers of theneuronal constructs (arrows). (H) Glial cells (GFAP, red) formconnections to capillary-like tubules (CD31, green) through apparentendfeet (arrows). (I) Microglia (IBA1, red) adopt ramified morphologiesand incorporate into vascular networks (CD31, green) (arrow). (J)Microglia (IBA1, white), glial cells (GFAP, red), and endothelial cells(CD31, green) for consecutive z-slices to illustrate depth. Endothelialcells and mesenchymal cells were added to the neuronal constructs at day9 and microglia were added at day 13. Scale bars: (A,D) 1000 μm;(B,C,E,F) Image size 1000×1000 μm.

FIGS. 8A-8D present images of microglia differentiation induced inthree-dimensional constructs. (A-D) Microglia (IBA1, red), glial cells(GFAP, white), and endothelial cells (CD31, green). (A) Full neuronalconstruct. (B) Reconstructed Z-stack illustrating boxed region in (A).(C, D) Maximum projection Z-stacks for boxed regions in (A) to showupper and lower regions of the neuronal construct. Scale bars: (A, B)1000 μm, (C) (D).

FIG. 9 is a FACS plot demonstrating microglia and macrophage precursor(MG) differentiation. Both adherent and nonadherent populations will beCD45⁺ (right-hand quadrants), but non-adherent cells will beCD14^(Low/Negative) (lower left quadrant) and adherent cells will beCD11b⁺/CD14⁺ (upper right quadrant; “MG”).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on the Inventors'discovery of methods for directing the differentiation of humanpluripotent stem cells into microglia, a type of glial cell that are theresident macrophages of the central nervous system (CNS). Microglia actas the first and main form of active immune defense in the CNS.Microglia have been found to be the primary source of brain cytokinesand have been implicated in neuronal pathologies associated with stroke,chronic neuroinflammation (e.g., Alzheimer's disease), and traumaticbrain injury. For review, see Giulian et al., J. Neurosci.15(11):7712-7726 (1995). Long thought to be derived from bone marrow,microglia have recently been shown to be derived from primitivemacrophages in the earliest wave of hematopoiesis in the yolk sac. SeeGinhoux et al., Science 330(6005):841-5 (2010). Prior to the Inventors'discoveries, there were no known protocols for deriving this cell typefrom human pluripotent stem cells. Moreover, obtaining and maintainingprimary microglia cultures is difficult. The Inventors discovered aprimitive, macrophage-like cell type of the hematopoietic lineage thathas the capability to develop ramified human microglia when added toneural tissues. Accordingly, the present invention provides methods ofefficiently and reproducibly producing and expanding human microgliasuitable for predictive analysis of candidate neurotoxic agents andother human tissue modelling applications. The present inventionprovides the first opportunity to study these cells in the properenvironment in an in vitro human model.

Methods

In exemplary embodiments, the methods provided herein comprisedifferentiating human pluripotent stem cells under conditions thatpromote differentiation of the pluripotent stem cells into hematopoieticprecursor cells, primitive macrophages, and microglia. Generally, thesecell types are identified by their surface phenotype, by the ability torespond to growth factors, and being able to differentiate in vivo or invitro into particular cell lineages. As used herein, the terms“hematopoietic precursor cells (HPCs)” and “hematopoietic progenitors”refer to immature progenitor cells of the hematopoietic lineage. HPCsare characterized by surface expression of CD45 and, in some cases,CD34, and a capacity to differentiate into myeloid progenitors. HPCs arealso known as hematovascular mesoderm progenitors. As used herein,“myeloid progenitors” are cells capable of differentiating into celltypes of the myeloid lineages. As used herein, the term “primitivemacrophages” refers to myeloid cells derived from HPCs and characterizedby CD45 expression and low or no expression of CD34. Primitivemacrophages are also characterized by expression of cell surface markersCD14 and CD11b, but show variable expression of CD68 (a marker of moremature macrophages) and do not express Iba-1 at a level that isdetectable by, for example, flow cytometry. Human pluripotent stemcell-derived primitive macrophages obtained according to a methodprovided herein can be cultured under particular culture conditions andcontacted to a synthetic or engineered construct comprising neurons andglia for maturation into ramified microglia.

In a first aspect, a method of obtaining HPCs comprises culturing humanpluripotent stem cells in a chemically defined culture medium undernormoxic conditions (i.e., where oxygen is provided at or about standardatmospheric levels) for about 24 hours. Preferably, the pluripotent stemcells are cultured in a chemically-defined basal culture mediumformulation comprising the defined components of culture medium “DF3 S”as set forth in Chen et al., Nature Methods 8:424-429 (2011), which isincorporated by reference herein as if set forth in its entirety. Asused herein, the terms “E7 culture medium” and “E7” are usedinterchangeably and refer to a chemically defined culture mediumcomprising or consisting essentially of DF3S supplemented to furthercomprise insulin (20 μg/mL), transferrin (10.67 ng/mL) and humanFibroblast Growth Factor 2 (FGF2) (100 ng/mL).

As used herein, the terms “E8 culture medium” and “E8” are usedinterchangeably and refer to a chemically defined culture mediumcomprising or consisting essentially of DF3S supplemented by theaddition of insulin, transferrin, human FGF2 (100 ng/mL), and humanTGFβ1 (Transforming Growth Factor Beta 1). As used herein, “E8 medium”refers to the chemically defined culture medium having the followingdefined components: DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64mg/L), sodium selenium (14 μg/L), and NaHCO₃ (543 mg/L), transferrin(10.7 mg/L), insulin (20 mg/L), FGF2 (100 m/L) and TGFβ1 (2 μg/L).Normoxic conditions are oxygen conditions of about 15% to about 20%oxygen (e.g., about 15%, 16%, 17%, 18%, 19%, 20% O₂). In some cases, thechemically defined culture medium additionally comprises a Rho kinaseinhibitor (ROCK inhibitor). ROCK inhibitors can be selected from thegroup consisting of Y-27632, Blebbistatin (a selective and high-affinitysmall molecule inhibitor of myosin heavy chain ATPase), and HA1077(fasudil). For example, a chemically defined culture medium appropriatefor use according to the methods described herein can be E8 mediumsupplemented with ROCK inhibitor Y-27632.

According to this differentiation protocol, pluripotent stem cells arecultured through the HPC differentiation step on a substrate thatpromotes cell adhesion. In exemplary embodiments, the substratecomprises Tenascin-C (“TenC”). TenC is expressed by mesenchymal cellsunderlying hematopoietic clusters in the aorta-gonado-mesonephros regionand is required for intraembryonic and postnatal hematopoiesis (Marshallet al., Dev. Dyn. 1999; 215:139-147; Nakamura-Ishizu et al., Blood 2012,119:5429-5437; Ohta et al., Blood. 1998; 91:4074-4083). Recombinanthuman TenC is commercially available from EMD Millipore. Afterdifferentiation of the pluripotent stem cells into HPCs on a TenC-coatedsurface, subsequent differentiation steps can take place on one or moreof the following surfaces: a non-adherent surface, a substratecomprising TenC, a substrate comprising a recombinant human vitronectinpolypeptide or fragment or variant thereof, or a self-coating substratesuch as Synthemax® (Corning), or a combination thereof.

For directed differentiation of the hematopoietic progenitor cells intomyeloid progenitors, the pluripotent stem cell-derived HPCs are culturedabout 3 days to about 5 days in a chemically defined, xeno-free culturemedium comprising FGF2, VEGF, thrombopoietin (TPO), stem cell factor(SCF), interleukin-6 (IL-6), and interleukin-3 (IL-3), where thehematopoietic progenitor cells differentiate into myeloid progenitors.

For directed differentiation of the myeloid progenitors in primitivemacrophages, the myeloid progenitors are cultured in the presence of achemically defined culture medium comprising insulin and granulocytemacrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine,whereby the cultured myeloid progenitors differentiate into a cellpopulation comprising at least 80% CD45⁺/CD11b⁺/CD14⁺ primitivemacrophages. Recombinant human GM-CSF and related cytokines arecommercially available.

In another aspect, a method of directing differentiation of humanpluripotent stem cells into HPCs comprises culturing the pluripotentstem cells under hypoxic conditions (i.e., where oxygen is provided at alevel lower than atmospheric) for about 40 hours to about 48 hours in achemically defined culture medium comprising one or more of thefollowing factors: a ROCK inhibitor (e.g., Y-27632), bone morphogeneticprotein 4 (BMP4), Activin A, and an inhibitor of glycogen synthase 3(GSK3). Preferably, the GSK3 inhibitor is selected from CHIR99021(6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile),lithium chloride (LiCl), and 6-bromoindirubin-3′-oxime (BIO). Hypoxicconditions are characterized by an oxygen concentration less than about10%. Preferably, hypoxic conditions are characterized by an oxygenconcentration of about 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2%. In exemplary embodiments,cells are cultured under hypoxic conditions of about 5% O₂ or lower(e.g., about 5%, about 4%, about 3%, about 2%, about 1%). The humanpluripotent stem cells can be cultured on any appropriate surface (e.g.,two-dimensional or three-dimensional).

According to this differentiation protocol, pluripotent stem cells arecultured through the HPC differentiation step on a substrate comprisingvitronectin polypeptides or fragments or variants thereof. In exemplaryembodiments, human pluripotent stem cells are cultured on avitronectin-coated surface for directed differentiation at a celldensity between about 2×10⁵ cells per cm² and about 2.5×10⁵ cells percm². After differentiation of the pluripotent stem cells into HPCs,subsequent differentiation steps (as set forth below) can take place ona vitronectin-coated surface, a non-adherent surface, or a TenC-coatedsurface.

In some cases, the method comprises further culturing the culturedpluripotent stem cells (cultured as described above) under hypoxicconditions in a culture medium comprising a Fibroblast Growth Factor(FGF), a vascular endothelial growth factor (VEGF), and an inhibitor ofTGFβ-mediated signaling, where culturing occurs for a length of timesufficient for the cultured pluripotent stem cells to differentiate intohematopoietic precursor cells. In some cases, the VEGF is VEGF-A or anisoform thereof. In some cases, the FGF is FGF2. The inhibitor ofTGFβ-mediated signaling can be selected from the group consisting ofSB-431542, a selective inhibitor of the activin receptor-like kinasereceptors ALK5, ALK4, and ALK7; and A-83-01.

Methods of the present invention further comprise directingdifferentiation of pluripotent stem cell-derived HPCs into progenitorsof the myeloid lineages (i.e., granulocyte, macrophage, erythroid, andmegakaryocyte) from pluripotent stem cell-derived hematopoieticprecursor cells. In humans, common myeloid progenitors (CMPs), which areprogenitor cells committed to the myeloid lineages, express CD34 andIL-3 R alpha (CD123). In exemplary embodiments, a method of obtainingmyeloid progenitors comprises culturing pluripotent stem cell-derivedhematopoietic precursor cells under normoxic conditions (i.e.,atmospheric oxygen levels) in a chemically defined growth mediumcomprising or consisting essentially of a FGF, a VEGF, thrombopoietin(TPO), and at least one cytokine selected from SCF, IL-6, and IL-3, or amixture thereof. In some cases, the VEGF is VEGF-A or an isoformthereof. In some cases, the FGF is FGF2.

Preferably, the method further comprises culturing the culturedpluripotent stem cell-derived HPCs under normoxic conditions in amyeloid differentiation culture medium. In exemplary embodiments, amyeloid differentiation culture medium is a chemically defined growthmedium comprising one or more hematopoietic cytokines such asgranulocyte-macrophage colony stimulating factor (GM-CSF; also known ascolony stimulating factor 2 (CSF2)), interleukin-3 (IL-3), orinterleukin-5 (IL-5). These cytokines are members of a discrete familyof cytokines that regulates the growth, differentiation, migration andeffector function activities of many hematopoietic cells and immunocytes(Broughton et al., Immunological Rev. 250(1):277-302 (2012)).

Cells are cultured in the myeloid differentiation medium until at leastabout 80% (e.g., at least 80%, 85%, 90%, 95%, 99%) of the resulting cellpopulation are CD45⁺/CD11b⁺/CD14⁺ myeloid progenitor cells that expresslittle, if any, CD34. Generally, myeloid progenitor cells arecharacterized by their expression of cell surface markers. For severalof these markers, the expression will be low or intermediate in level.While it is commonplace to refer to cells as “positive” or “negative”for a particular marker, actual expression levels are a quantitativetrait. The number of molecules on the cell surface can vary by severallogs, yet still be characterized as “positive.” Accordingly,characterization of the level of staining permits subtle distinctionsbetween cell populations. Expression levels can be detected or monitoredby flow cytometry, where lasers detect the quantitative levels offluorochrome (which is proportional to the amount of cell surfaceantigen bound by the antibodies). Flow cytometry orfluorescence-activated cell sorting (FACS) can be used to separate cellpopulations based on the intensity of antibody staining, as well asother parameters such as cell size and light scatter. Although theabsolute level of staining may differ with a particular fluorochrome andantibody preparation, the data can be normalized to a control.

A loss of or reduction in CD34 expression is indicative of a transitionfrom a hematopoietic stem cell (HSC)-like progenitor cell to a myeloidprogenitor cell: a CD45⁺/CD11b⁺/CD14⁺ primitive macrophage. Primitivemacrophages exhibit variable expression of CD68, which is a biologicalmarker of more mature macrophages, and do not express a detectable levelof Iba-1.

In exemplary embodiments, primitive macrophages derived according to themethods described herein are further cultured for a minimum of five daysin a macrophage differentiation medium comprising Iscove's ModifiedDulbecco's Media (IMDM), interleukin-1 beta (IL-1β), and a hematopoieticgrowth factor such as macrophage colony-stimulating factor (M-CSF),which is also known as colony stimulating factor 1 (CSF1). In somecases, primitive macrophages are cultured in the presence of macrophagedifferentiation medium for about 5 days and, preferably, contacted to athree-dimensional (3D) neural tissue construct comprising human neuronsand glia, whereby the contacted primitive macrophages mature intoramified (“resting”) microglia. For example, primitive macrophagescultured in the presence of macrophage differentiation medium for about5 days are contacted or introduced to a 3D hydrogel-based neural tissueconstruct as described in U.S. application Ser. No. 14/986,382 and U.S.application Ser. No. 14/986,363 respectively, which are incorporatedherein as if set forth in their entirety.

Any appropriate method can be used to detect expression of biologicalmarkers characteristic of cell types described herein. For example, thepresence or absence of one or more biological markers can be detectedusing, for example, RNA sequencing, immunohistochemistry, polymerasechain reaction, qRT-PCR, or other technique that detects or measuresgene expression. In exemplary embodiments, a cell population obtainedaccording to a method provided herein is evaluated for expression (orthe absence thereof) of biological markers of mature microglia such asIba-1. Myeloid markers and macrophage associated markers include, forexample, CD14, CD16, CSFR-1, CD11b, CD206 (also known as macrophagemannose receptor or MMR), CD68, and CD163. Quantitative methods forevaluating expression of markers at the protein level in cellpopulations are also known in the art. For example, flow cytometry isused to determine the fraction of cells in a given cell population thatexpress or do not express biological markers of interest. Biologicalmarkers for perivascular cells and microglia include antibodies havingspecificity to CD45, CD68, or HLA-DR complex.

As used herein, “pluripotent stem cells” appropriate for use accordingto a method of the invention are cells having the capacity todifferentiate into cells of all three germ layers. Suitable pluripotentcells for use herein include human embryonic stem cells (hESCs) andhuman induced pluripotent stem (iPS) cells. As used herein, “embryonicstem cells” or “ESCs” mean a pluripotent cell or population ofpluripotent cells derived from an inner cell mass of a blastocyst. SeeThomson et al., Science 282:1145-1147 (1998). These cells express Oct-4,SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and appear as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleolus. ESCsare commercially available from sources such as WiCell ResearchInstitute (Madison, Wis.). As used herein, “induced pluripotent stemcells” or “iPS cells” mean a pluripotent cell or population ofpluripotent cells that may vary with respect to their differentiatedsomatic cell of origin, that may vary with respect to a specific set ofpotency-determining factors and that may vary with respect to cultureconditions used to isolate them, but nonetheless are substantiallygenetically identical to their respective differentiated somatic cell oforigin and display characteristics similar to higher potency cells, suchas ESCs, as described herein. See, e.g., Yu et al., Science318:1917-1920 (2007).

Induced pluripotent stem cells exhibit morphological properties (e.g.,round shape, large nucleoli and scant cytoplasm) and growth properties(e.g., doubling time of about seventeen to eighteen hours) akin to ESCs.In addition, iPS cells express pluripotent cell-specific markers (e.g.,Oct-4, SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, but not SSEA-1). Inducedpluripotent stem cells, however, are not immediately derived fromembryos. As used herein, “not immediately derived from embryos” meansthat the starting cell type for producing iPS cells is a non-pluripotentcell, such as a multipotent cell or terminally differentiated cell, suchas somatic cells obtained from a post-natal individual.

Human iPS cells can be used according to a method described herein toobtain primitive macrophages and microglial cells having the geneticcomplement of a particular human subject. For example, it may beadvantageous to obtain microglia that exhibit one or more specificphenotypes associated with or resulting from a particular disease ordisorder of the particular mammalian subject. In such cases, iPS cellsare obtained by reprogramming a somatic cell of a particular humansubject according to methods known in the art. See, for example, Yu etal., Science 324(5928):797-801 (2009); Chen et al., Nat. Methods8(5):424-9 (2011); Ebert et al., Nature 457(7227):277-80 (2009); Howdenet al., Proc. Natl. Acad. Sci. U.S.A. 108(16):6537-42 (2011). Inducedpluripotent stem cell-derived microglial cells allow modeling of drugresponses in tissue constructs that recapitulate neural or other tissuein an individual having, for example, a particular disease. Even thesafest drugs may cause adverse reactions in certain individuals with aspecific genetic background or environmental history. Accordingly, humansubject specific iPS cell-derived primitive macrophages and microgliaare useful to identify genetic factors and epigenetic influences thatcontribute to variable drug responses.

Subject-specific somatic cells for reprogramming into inducedpluripotent stem cells can be obtained or isolated from a target tissueof interest by biopsy or other tissue sampling methods. In some cases,subject-specific cells are manipulated in vitro prior to use in athree-dimensional hydrogel-based tissue construct of the invention. Forexample, subject-specific cells can be expanded, differentiated,genetically modified, contacted to polypeptides, nucleic acids, or otherfactors, cryo-preserved, or otherwise modified prior to introduction toa three-dimensional tissue construct.

In exemplary embodiments, human pluripotent stem cells are cultured in achemically-defined basal culture medium formulation comprising thedefined components of culture medium “DF3S” as set forth in Chen et al.,Nature Methods 8:424-429 (2011), which is incorporated by referenceherein as if set forth in its entirety. DF3S medium: DMEM/F12,L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14μg/L), and NaHCO₃ (543 mg/L). Preferably, human pluripotent stem cells(e.g., human ESCs or iPS cells) are cultured in the absence of a feederlayer (e.g., a fibroblast layer), a conditioned medium, or a culturemedium comprising poorly defined or undefined components. As usedherein, the terms “chemically defined medium” and “chemically definedcultured medium” also refer to a culture medium containing formulationsof fully disclosed or identifiable ingredients, the precise quantitiesof which are known or identifiable and can be controlled individually.As such, a culture medium is not chemically defined if (1) the chemicaland structural identity of all medium ingredients is not known, (2) themedium contains unknown quantities of any ingredients, or (3) both.Standardizing culture conditions by using a chemically defined culturemedium minimizes the potential for lot-to-lot or batch-to-batchvariations in materials to which the cells are exposed during cellculture. Accordingly, the effects of various differentiation factors aremore predictable when added to cells and tissues cultured underchemically defined conditions. As used herein, the term “serum-free”refers to cell culture materials that are free of serum obtained fromanimal (e.g., fetal bovine) blood. In general, culturing cells ortissues in the absence of animal-derived materials (i.e., underxenogen-free conditions) reduces or eliminates the potential forcross-species viral or prion transmission.

In a further aspect, provided herein is a method of screening a compoundfor toxicity. In exemplary embodiments, the method comprises exposing atest compound to a composition comprising human microglial cells andassaying for a toxic effect of the compound on one or more aspects ofhuman microglial growth or development. Preferably, the composition isobtained by contacting human pluripotent stem cell-derived primitivemacrophages to a chemically defined, xenogen-free three-dimensional (3D)tissue construct that includes stratified layers of human neurons andglia. Upon addition of primitive macrophages to such a 3D composition,the primitive macrophages differentiate to form a composition comprisinghuman microglial cells.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described herein.

As used herein, “a medium consisting essentially of” means a medium thatcontains the specified ingredients and those that do not materiallyaffect its basic characteristics.

As used herein, “effective amount” means an amount of an agentsufficient to evoke a specified cellular effect according to the presentinvention.

As used herein, “about” means within 5% of a stated concentration range,density, temperature, or time frame.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples. It is specifically contemplated thatthe methods disclosed are suited for pluripotent stem cells generally.All papers and patents disclosed herein are hereby incorporated byreference as if set forth in their entirety.

EXAMPLES Example 1 Protocol for Directed Differentiation of hESCs intoHematopoietic Precursor Cells

The first stage of differentiation requires the derivation ofhematopoietic precursor cells (HPCs) from human pluripotent stem cells.The following protocol was developed and validated for obtaininghemogenic endothelium that ultimately gives rise to HPCs. Tenascin C(TenC), an extracellular matrix protein associated with HSC niches,strongly promotes HE and definitive hematopoiesis in this system.

Microglia/macrophage precursors were produced using feeder-free,chemically defined conditions by modifying a previous protocol fordifferentiating H1 ES cells down mesendoderm and hemogenic endotheliumlineages (see Slukvin 2006; Vodyanik et al., Blood (2006)108:2095-2105). 6-well plates were first coated with 40 μg Tenascin Covernight 4° C. Tenascin C plates were rinsed with PBS, and then seededwith singularized H1 ES cells at a density of 62,500 cells/cm² in E8medium+10 μM ROCK inhibitor Y-27632. Directed differentiation of thehuman pluripotent stem cells occurs following culture of the cells in E8medium+ROCK inhibitor for about 24 hours under normoxic conditions.

E8 Medium: DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/L),sodium selenium (14 μg/L), and NaHCO₃ (543 mg/L), transferrin (10.7mg/L), insulin (20 mg/L), FGF2 (100 μg/L) and TGFβ1 (2 μg/L).

Example 2 Alternative Protocol for Directed Differentiation of hESCsinto Hematopoietic Precursor Cells and Primitive Macrophages

Provided below is an alternative protocol for obtaining HPCs andprimitive macrophages from human pluripotent stem cells.

TABLE 1 Differentiation Base Medium Add: Final Concentration Definedcomponent: Iscove's Modified Dulbecco's 500 mL 1X Media (IMDM) + F12L-ascorbic acid 2-phosphate 32 mg Mg2⁺ salt Monothioglycerol 20 uLSodium selenite 6 μL 0.7 mg/mL Polyvinyl alcohol 20 mg/L GlutaMAX ™ 100Xsupplement 5 mL (Gibco ®) Non-Essential Amino Acids Solution 5 mL (NEAA)(100X) (Gibco ®) Chemically Defined Lipid Concen- 5 mL trate (CDLC) 100X(Gibco ®) Base Cytokines Insulin 10 mg/mL Holo-Transferrin 10.6 mg/mL

TABLE 2 Differentiation Medium 1 (DM1) Defined component: (Cytokines 12mL) Add: Final Concentration: Base media  12 mL 1X FGF2 (100 μg/mL)  6μL 50 ng/mL BMP4 (100 μg/mL)  6 μL 50 ng/mL Activin A (10 μg/mL) 15 μL12.5 ng/mL LiCl (2M) 12 μL 2 mM

TABLE 3 Differentiation Medium 2 (DM2) Defined component: (Cytokines 12mL) Add: Final Concentration: Base media 12 mL  1X FGF2 (100 μg/mL) 6 μL50 ng/mL VEGF (100 μg/mL) 6 μL 50 ng/mL SB-431542 10 μM

TABLE 4 Differentiation Medium 3 (DM3) Defined component: Add: FinalConcentration: Base media 12 mL  1X FGF2 (100 μg/mL) 6 μL 50 ng/mL VEGF(100 μg/mL) 6 μL 50 ng/mL TPO (100 μg/mL) 6 μL 50 ng/mL SCF (100 μg/mL)6 μL 50 ng/mL IL-6 (100 μg/mL) 6 μL 50 ng/mL IL-3 (10 μg/mL) 12 μL  10ng/mL

TABLE 5 Differentiation Medium 4 (DM4) Defined component: Add: FinalConcentration: Base media 12 mL 1X GM-CSF 200 ng/mL

TABLE 6 Differentiation Medium 5 (DM5) Defined component: FinalConcentration: Iscove's Modified Dulbecco's Media (IMDM) 1XHeat-inactivated Fetal Bovine Serum (FBS) 10% IL-1β 10 ng/mL M-CSF 20ng/mL

Initiate Early Mesoderm Differentiation: Twenty four hours after platingH1 ES cells, E8 media was aspirated and replaced with DM1+1 μM Y-27632.Cells were then cultured under hypoxic conditions (5% O₂) for two days(do not expose cells to normoxia) on vitronectin-coated plates. Duringthe two days of culture, cells will detach and reattach. Care is takento avoid disturbing the culture, as cells tend to aggregate in themiddle of the plate and affect differentiation efficiency.

HPC Differentiation and Expansion: On day 2, cultures were checked forsurviving cell clumps that have not fully reattached. If cells are stillfloating, use a 10 ml pipet to pull media off plate, centrifuge floatingcells and cell clumps @ 300×g to form a pellet, aspirate DM1 andresuspend in DM2, plate cells back into same plate, and continue culturein a hypoxic incubator. If only debris is present, aspirate DM1 and addDM2 slowly as to not disrupt the adherent cells, continue culture in ahypoxic incubator.

On day 4, DM2 medium was aspirated and replaced with DM3 medium. Culturewas continued in a normoxic incubator. On day 6 of culture (2 days afteradding DM3 media), additional DM3 media was added without aspiratingmedia already present. Culture was continued in a normoxic incubator.Cells were expanded for an additional 3-5 days in DM3 (or for a longertime if cells are not fully adherent after hematovasculardifferentiation). If media color indicates a significant drop in pH,half of the media volume was removed and placed in a low-attachmentdish. An additional volume of DM3 was added to both culture plates.After 3-5 days, spent media containing floating HPCs was collected andcentrifuged @300×g to pellet. The resulting cells are hematopoieticprecursor cells (also known as hematopoietic progenitors).

Myeloid Progenitor Differentiation: HPCs were cultured in myeloidprogenitor medium DM4, adding 1×10⁶ cells/ml to a low attachment culturedish (at this point, cells can be grown on a 10 cm dish) under normoxicconditions to direct differentiation of the HPCs into myeloidprogenitors. The myeloid progenitors were expanded for 2-5 days in DM4medium (at least 2 days required for proper transition to macrophages),adding media if pH significantly drops (half/half mixture; do nottransfer cells). Up to 2×10⁷ myeloid progenitors were obtained per 10 cmdish. During expansion in DM4 medium (2-5 days), floating cells werecollected for an optional sorting step to identify CD34⁺ and CD45⁺cells.

Microglia/Macrophage Precursor Differentiation: After 2-5 days ofmyeloid progenitor expansion, 5×10⁵ floating cells were added tomacrophage differentiation medium DM5 in a 10 cm treated tissue culturedish. Cells were cultured for 3 days and then an equivalent volume ofDM5 medium (without aspiration) was added. After 5 days (meaning,following 2 additional days in DM5), ˜50-70% of cells will haveattached. When cells reach ˜70-80% confluence (adherent cells), theremaining floating cells were transferred to a new 10 cm dish to promoteadhesion. FACS was performed to confirm that the resulting cellsare >80% CD11b⁺, CD45⁺, and CD14⁺. See FIGS. 4A-6D. Floating cells areCD14^(Low/Negative) but will continue to mature. On days 5-10, cellsbegin to attach. These cells (as well as some floating cells) should beCD11b⁺ and CD14⁺ (˜60-90%). Culture in DM5 medium was continued.

Identification of Cell Types During Directed Differentiation: HPCs wereidentified based on expression of CD45 (greater than 75%) with aproportion of cells co-expressing CD34. Myeloid progenitors were derivedand expanded from this cell population. Cells expressing CD45 (greaterthan 85%) with little or no CD34 expression, were identified asprimitive macrophages. The primitive macrophages also expressed cellsurface markers CD14 and CD11b, but showed variable expression of themore mature microglial marker CD68 and did not express Iba-1 at adetectable level as assayed using flow cytometry. See FIGS. 4A-6D.Primitive macrophages were matured for a minimum of five days in amaturation culture medium and were then added to neural tissues tocomplete the maturation to ramified (mature, resting) microglia.

Assaying for Phagocytosis by Microglia/Macrophage Precursors: Aliquotsof zymosan A S. cerevisiae BioParticles® (Texas Red® conjugate; LifeTechnologies) were prepared in PBS. Approximately 5×10⁶ particles in 500μL PBS were added to each well of a E-well plate containing ˜400-500Kmicroglia/macrophage precursors in DM5 media. Phagocytosis was imagedover a 24 hour time period (images captured every 10 min.) using a NikonBiostation® CT.

Example 3 Characterizing Human Pluripotent Stem Cell-Derived PrimitiveMacrophages

To demonstrate that human primitive macrophages obtained according tothe methods provided herein are structurally and functionally comparableto primary cultures, fluorescently tagged, inactivated yeast particleswere added to in vitro cultures to induce phagocytosis. Primitivemacrophages completely cleared the cell culture medium of tagged yeastparticles within 24 hours of induction. Human embryonic stem cells,which served as a control, did not phagocytize the yeast particles.

To demonstrate that the primitive macrophage cell populations did notcomprise microglia, the cells were fixed and stained with afluorescently tagged antibody specific to Iba-1, a hallmark microglialtranscription factor. No Iba-I⁺ cells were observed, indicating thatnone of the pluripotent stem cell-derived cells had differentiated intomicroglia.

During the formation of the human cerebral cortex, Iba1⁺ microglia beginto populate the cortical primordial plexiform layer, or preplate (atransient structure comprising the marginal zone and the subplate thatarises during cortical development), as early as 5.5 gestational weeks(GW), but they only minimally interact with blood vessels that havepenetrated the human cortex before about 9 GW. To mimic the in vivorecruitment of blood vessels and microglia after the initial formationof the neural tube and the developmental timing when Iba1⁺ microgliabegin to interact with capillaries of the cortex, primitive macrophageswere added to a pre-formed hydrogel neural tissue construct completewith embedded vasculature as described in U.S. application Ser. No.14/986,363, which is incorporated herein as if set forth in itsentirety. Primitive macrophages were added to hydrogel neural tissueconstructs after initial vascular network organization and after neuralprogenitor cells had self-assembled into multilayered structures withradially organized neural and glial populations reminiscent of the earlyneuroepithelium. By day 21 of differentiation on neural tissueconstructs, the constructs contained an extensive neural network, cellsexhibiting neural and glial phenotypes, interconnected capillarynetworks, and microglia-like cells. RNA-Seq was used to quantitativelyassess sample uniformity by comparing differential gene expression forreplicate neural constructs after 14 and 21 days of differentiation onhydrogels. In addition, Iba-I protein expression was detected byfluorescent antibody staining. Replicate samples were characterized bySpearman's correlation coefficients (ρ)≥0.99 to at least 21 days ofdifferentiation. See Table 7 and FIGS. 3A-3D. RNA sequencing (RNA-seq)revealed an increase in expression of CD68, a microglial cell marker.RNA-seq also identified several characteristic microglia genes that weredetectable only when primitive macrophages/microglia precursor cellswere incorporated into the neural constructs, such as CD11B (ITGAM),TREM2, and IBA1 (AIF1). Iba1⁺ cells were distributed throughout theneural constructs by day 21 (FIGS. 2A-2G), and adopted ramifiedmorphologies, which is a distinguishing feature for microglia in theresting state. Iba1⁺ cells associated with endothelial tubules (FIGS.2A-2G, 3A-3D, 7A-7J, and 8A-8D), which has been observed during humandevelopment and suggests a possible role for microglia in guidingvascular organization within the neural constructs. Therefore, human EScell-derived primitive macrophages exhibit several properties consistentwith a microglia-like phenotype observed within the neural constructs.These data demonstrate that three-dimensional multilayered neuraltissue-like constructs can be produced with remarkable uniformity whenES cell-derived precursor cells are cultured on bioactive hydrogels.

FIG. 9 presents FACS data for Microglia and macrophage precursor (MG)differentiation. Both adherent and nonadherent populations are CD45⁺,but non-adherent cells are CD14^(Low/Negative) and adherent cells areCD11b⁺CD14⁺. On days 5-10, non-adherent cells began to attach anddifferentiate into CD11b⁺ and CD14⁺ cells.

TABLE 7 Gene Expression in Neural Constructs Average Standard DeviationGenes Day 16 Day 21 Day 16 Day 21 AIF1/IBA1 12.1 15.6 3.3 8.8 ITGAM/CD2.3 1.2 0.6 1.2 PTPRC 4.1 3.6 0.9 3.3 CX3CR1 3.8 3.7 0.8 4.6 CD68 17.323.8 4.0 18.9 CD14 1.8 3.0 1.6 1.4 Normalized expression (TPM; N = 4)

Materials and Methods—Neural Tissue Constructs Comprising PluripotentStem Cell-Derived Microglia/Macrophage Precursors

Hydrogel Polymerization: Polyethylene glycol (PEG) hydrogels were formedusing thiol-ene photopolymerization chemistry, with modifications frompreviously a published protocol (Fairbanks 2009). Stock solutions of8-arm PEG-norbornene (20000 MW, JenKem USA, 8ARM (TP)-NB-20K) wereprepared at a final concentration of 300 mg/mL by dissolving 300 mg ofsolid/0.8 mL PBS to account for volume occupied by 8-arm PEG-norbornenesolid, sterile filtered through a 0.2 μm nylon syringe filter (Fisher),and stored as frozen aliquots. Matrix metalloproteinase (MMP)-degradablePEG hydrogels were formed using an amino acid sequence modified from anative collagen sequence (Nagase 1996) (KCGPQG˜IWGQCK (SEQ ID NO:1);Active sequence in bold, cleave site=(˜); Genscript, >90% purity,C-terminus amidated), with cysteines on each end to crosslink 8-armPEG-norbornene molecules. Cell adhesion was promoted by incorporatingCRGDS peptide (SEQ ID NO:2) (2 mM final monomer solution concentration;Genscript, >90% purity, C-terminus amidated), an amino acid sequencederived from fibronectin (Pierschbacher 1984). Stock MMP-peptide (˜75 mMpeptide/150 mM SH) and CRGDS peptide (˜100 mM) solutions were preparedand sterile filtered through a 0.22 μm low protein bindingpolyvinylidene difluoride (PVDF) syringe filter (Millex) and the finalconcentration was verified after filtration using an Elman's assay(Thermo Scientific; modification of Manufacturier's protocol: PBS usedto dissolve all reagents).

The final monomer formulation for PEG hydrogels was 40 mg/mL 8-armPEG-NB, 4.8 mM MMP-peptide crosslinker (9.6 mM cysteines, 60% molarratio relative to norbornene arms), 2 mM CRGDS (SEQ ID NO:2), and 0.05%(wt/wt) Irgacure® 2959 photoinitiator (BASF Schweiz AG, Basel,Switzerland). Hydrogels were formed by pipetting 30 μL monomer into24-well BD Transwell inserts (1 μm pores, Fisher; Quality controlexperiments) or 40 μL into Corning HTS Transwell-24 well permeablesupport (0.4 μm pores, Sigma Aldrich; Toxicity experiments). Afterpipetting, any gaps between the PEG monomer solution and the edge of theinsert (due to surface tension) were removed by tilting the insert plateand gently tapping until the solution uniformly covered the bottom ofthe transwell insert membrane. Transwell plates containing inserts andmonomer solutions were placed on the top shelf of a UVP XX-15 lamp stand(Fisher) and exposed to ˜365 nm centered UV light (UVP XX-15L lamp,Fisher) for 2.5 minutes. After polymerization, hydrogels were incubatedin DF3S medium overnight to allow swelling and equilibration (5% CO₂,37° C.).

Seeding H1 ES Cell-Derived Neural Progenitor Cells (NPCs) on PEGHydrogels: Cryopreserved NPCs were thawed and expanded on 6-well platescoated with Matrigel® (BD Biosciences, 0.5 mg per plate for at least 1hour) and cultured in neural expansion medium. One vial of frozen NPCs(˜1.2×10⁷ cells) was thawed and plated in 3 wells of a Matrigel® coated6-well plate (2 vials were thawed in one Matrigel® coated 10 cm dish),cultured for 2-3 days (depending on initial confluence) and passaged 1:3using Accutase™. NPCs were passaged 1:3 after 2 days of additionalculture, expanded for 2-3 more days and used for experiments.

NPCs were removed from the plate using 1 mL Accutase/well, from which analiquot was removed for counting. After adding the appropriate volume ofcell suspension to a conical vial, NPCs were pelleted at 0.2 G for 4minutes. NPCs were resuspended and seeded in neural expansion medium ata density of 100,000 cells/24-well insert. NPCs were allowed to attachovernight, and then neural expansion medium was exchanged on Day 1 andevery 2 days for the remainder of the experiment. For each mediumexchange, all medium under the insert was aspirated, while approximately¾ of the medium was removed from the top by sliding the pipette tip downthe side of the well to avoid damaging the developing neural tissueconstructs.

Differentiation and Growth on PEG Hydrogels of H1 ES Cell-DerivedEndothelial Cells (ECs), Mesenchymal Stem Cells (MSCs), andMicroglia/Macrophage Precursors (MG): Endothelial cells were expandedfrom cryopreserved stocks on fibronectin-coated plates (LifeTechnologies, 100 μg per plate) using E7BV media, with one vial (˜1×10⁶cells) per 6 wells of a 6-well plate or a single 10 cm dish. ECs weresplit 1:3 after 2 days using Accutase, cultured for an additional 3days, and then used for experiments.

E8BA medium: E8 supplemented with BMP4 (5 μg/L) and Activin A (25 μg/L).E7V medium: E8 minus TGFβ1, supplemented with VEGF-A (50 μg/L). E7BVimedium: E7V supplemented with BMP4 (50 μg/L) and SB431542 (5 μM, TGFβinhibitor) (Inman 2002). E7BV medium: E7V supplemented with BMP4 (50μg/L).

At day 9, endothelial cells (ECs) and MSCs were seeded on top of thedifferentiating NPC layer at a total density of 100,000 cells/well, witha 5:1 ratio of ECs:MSCs (83.3K:16.7K) (FIGS. 1A-1B). Both ECs and MSCswere harvested using Accutase and counted before centrifugation. Cellswere counted and mixed in the appropriate ratio, centrifuged, andresuspended for seeding. Neural expansion medium was exchanged on day 11(2 days after seeding ECs and MSCs). At day 13, microglia/macrophageprecursors were harvested and seeded at a density of 100,000cells/insert (FIGS. 1A-1B). Neural expansion medium was exchanged on day14, and then every other day until samples were collected for RNA,sorting, or immunofluorescence imaging.

Human ES Cell Differentiation into Microglia/Macrophage Precursors (MG):

Microglia/macrophage precursors were produced using feeder-freeconditions by modifying a previous protocol for differentiating H1 EScells down mesendoderm and hemogenic endothelium lineages (see Uenishiet al. (2014) Stem Cell Rep 3(6):1073-1084). E-well plates were firstcoated with 40 μg Tenascin C overnight at 4° C. Tenascin C plates wererinsed with PBS, and then seeded with singularized H1 ES cells at adensity of 62,500 cells/cm² in E8 medium+10 μM Y-27632 (ROCK inhibitor,R&D Systems). Cells were cultured for 24 hours under normoxicconditions.

Initiate Early Mesoderm Differentiation. 24 hours after plating H1 EScells, E8 media was aspirated and replaced with DM1+1 μM Y-27632. Cellswere then cultured under hypoxic conditions (5% O₂) for two days (do notexpose cells to normoxia). During the two days of culture, cells detachand reattach. It is important that the culture is not disturbed, ascells will aggregate in the middle of the plate, affectingdifferentiation efficiency.

Continue Hematovascular Mesoderm Differentiation. On day two, theculture was checked for surviving cell clumps that had not fullyreattached. If non-adherent cells were present, a 10 mL pipette tip wasused to gently pull media off plate, and the non-adherent cells and cellclumps were centrifuged at 300×g for five minutes to form a pellet. DM1was aspirated from the pellet, and the cells were resuspended in DM2.Cells were gently plated back into same plate, and culture was continuedin a hypoxic incubator. If only debris was present, DM1 was aspiratedand DM2 was added slowly as to not disrupt the adherent cells. Culturewas continued in a hypoxic incubator.

Differentiate and Expand Hemogenic Endothelial Cells into HematopoieticProgenitor Cells (HPCs). On day 4, DM2 medium was aspirated and replacedwith DM3 medium. Culture was continued under normoxic conditions. On day6 of culture (two days after adding DM3 media), additional DM3 media wasadded without aspirating media already present. Culture was continued ina normoxic incubator. Cell cultures were expanded for an additional 3-5days in DM3 (longer time is required when cells not fully adherent afterhematovascular differentiation). If media color indicated a significantpH drop, half of the media volume was removed from the plate and placedinto a low attachment dish. An additional volume of DM3 (1:1 mix of oldand fresh media) was added to both culture plates. After 3-5 days, spentmedia containing non-adherent HPCs was collected and centrifuged at300×g for about five minutes to pellet.

Myeloid Progenitor (MP) Differentiation. Expansion was continued inmyeloid progenitor medium DM4, where 1×10⁶ HPCs/mL were to a lowattachment culture dish under normoxic conditions. At this point, thecells could be grown in a 10 cm dish under normoxic conditions. Cellswere expanded for 2-5 days in the DM4 medium. At least five days inculture was required for proper transition to macrophages, but no morethan five days. DM4 was added if the culture's pH significantly dropped(half/half mixture; do not transfer cells). Up to 2×10⁷ cells wereobtained from a 10 cm dish. During expansion in DM4 medium (2-5 days),non-adherent cells were collected for sorting to identify CD34⁺ andCD45⁺ cells.

Microglia/Macrophage Precursor (MG) Differentiation. After 2-5 days ofmyeloid progenitor expansion, 5×10⁵ non-adherent cells were added tomacrophage differentiation medium DM5 in a 10 cm tissue culture treateddish. Cells were cultured for three days, then an equivalent volume ofDM5 media was added without aspiration of the media. After five days(two additional days in DM5), ˜50-70% of cells had attached. When cellsreached ˜70-80% confluence (adherent cells), remaining non-adherentcells were transferred to a new 10 cm dish to promote adhesion. As shownin FIG. 9, both adherent and non-adherent populations are CD45⁺, butnon-adherent cells will be CD14^(Low/Negative) and adherent cells willbe CD11b⁺/CD14⁺. On days 5-10, non-adherent cells began to attach anddifferentia into CD11b⁺ and CD14⁺ cells. Culture in DM5 medium wascontinued.

Toxicity Screening Experiments: For toxicity screening experiments,cells were seeded as described above, but with 65,000 cells/well forECs+MSCs (also 5:1 ratio) and 15,000 cells/well for microglia/macrophageprecursors. Neural constructs were treated with non-toxic or toxiccompounds starting at day 14, with medium exchanged every 2 days. Toxicchemicals were chosen based on previous literature support forneurotoxicity (Adams 1993; Cooper 1998; Crofton 2011; Eskes 2003;Grandjean 2014; Lidsky 2003; Radio 2010; Zurich 2002).

For the quality control assays, RNA was collected on days 14 and 21. Forthe 3D toxicity screening experiments, RNA was collected on days 16 and21 (permitting 2 days of chemical exposure before collecting at thefirst time point).

Immunofluorescence Imaging: Blocking buffer: 0.25% Triton X-100 and 1%BSA in PBS; Incubation buffer: 0.05% Triton X-100 and 1% BSA in PBS;Rinse buffer: 0.05% Triton X-100 in PBS.

Primary Antibodies: Rabbit anti-β3-tubulin (1:500; Cell Signaling, mAb#5568S), mouse anti-β3-tubulin (1:500; R&D Systems, MAB1195), rabbitanti-calretinin (1:100-1:200: Abcam, ab137878), rabbit anti-GABA (1:200:Abcam, ab43865), rabbit polyclonal fibrillary acidic protein (GFAP)(1:500; Dako, Z033401-2), goat anti-glial fibrillary acidic protein(GFAP) (1:100-1:200; C-19; sc-6170, Santa Cruz Biotechnology), mouseanti-phospho-vimentin (1:200; S55 [4A4]; Abcam, ab22651), mouseanti-CD31 (1:200; Endothelial Cell, Clone JC70A; DAKO, M082301-2), mouseanti-04 (1:100-1:200; clone 81; Millipore, MAB345), Chicken polyclonalanti-Tbr1 (1:100-1:200; Millipore, AB2261), mouse anti-SOX-2 (CellSignaling, mAb #4900S), rabbit anti-SOX-2 (Cell Signaling, mAb #3579S),mouse anti-MAP2, (clone AP20; Millipore, MAB3418), mouse anti-Reelin(1:100; clone G10, a.a. 164-496; Millipore, MAB5364), mouse anti-Brn-2(POU3F2) (1:200; clone 8C4.2; Millipore, MABD51), rabbit anti-Brn-2(POU3F2) (1:200; Cell Signaling, mAb #12137S), rabbit anti-Ctip2(Bcl-11b) (1:200; Cell Signaling, mAb #12120S), rabbit anti-VGLUT2(1:100; Abcam), mouse anti-MAP2 (1:500; clone AP20; Millipore, MAB3418),goat anti-Iba1 (1:100; Abcam, ab5076), rabbit anti-Tyrosine Hydroxylase(Cell Signaling, mAb ##27925), rabbit anti-PDGFR-α (1:100; Santa CruzBiotechnology, sc-338).

Secondary Antibodies: Alexa Fluor secondary antibodies were used for allexperiments (Life Technologies): Donkey anti-goat 568 (A11057) or 647(A21447); Donkey anti-rabbit 488 (A21206), 568 (A10042), or 647(A-31573); Donkey anti-mouse 488 (A-21202), 568 (A10037), or 647(A31571); Goat anti-chicken (A11041).

Immunostaining Full Neural Constructs: All steps for immunostaining wereperformed within transwell inserts. Neural constructs were fixed for 60min. using 2% buffered formalin and then rinsed with PBS (or stored at4° C. until immunostaining). Neural constructs were permeabilized andblocked in blocking buffer (at least 60 min.). For some experiments,blocking buffer was used for all steps until final rinse, with similarresults. Primary antibodies were prepared in incubation buffer, added tothe neural constructs, and incubated overnight at 4° C. Neuralconstructs were then rinsed (2× with rinse buffer, at least 60 min./ea.)followed by a third rinse step (blocking buffer, at least 60 min.).Secondary antibodies and 1:1000 DAPI (Sigma) were prepared in incubationbuffer, added to the neural constructs, and incubated overnight at 4° C.(or at least 4 hours at room temperature). Neural constructs were rinsed2×60 min. in rinse buffer, followed by an overnight rinse at 4° C. inincubation buffer. Samples were then stored in PBS until furtherprocessing (typically at least 24 hours).

Neural constructs were removed from the transwell insert by cutting thebottom edge of the membrane, separated from the membrane, and mounted inaqua polymount solution (Polysciences, Inc.) on the bottom of a 35 mmglass bottom dish (MatTek). To limit bubble formation in the mountingsolution, a thin layer was first added to the glass bottom of the 35 mmdish. The neural construct was usually placed face down into the layerof mounting solution (with some samples placed face up), after which adrop of mounting solution was added to cover the construct. A coverslipwas then dropped onto the neural construct in mounting solution andallowed to settle, rotating the dish to ensure uniform coverage of themounting solution under the coverslip. The coverslip was allowed tosettle overnight at 4° C., and sealed around the edges with fingernailsealant. The samples remained stable for imaging for at least 1 month.

Immunostaining Cryopreserved Sections: Neural constructs were fixed inthe transwell insert for 60 min. using 2% buffered formalin and rinsedwith PBS (overnight at 4° C.). The samples were then rinsed in 15%Sucrose/PBS (at least 24 hours, 4° C.) followed by 30% Sucrose/PBS (atleast 24 hours, 4° C.). Neural constructs were removed from thetranswell insert by cutting the bottom edge of the membrane, separatedfrom the membrane, and placed face down into cryogel (Tissue-Tekembedding medium), and stored frozen at −80° C. until furtherprocessing. Frozen samples were equilibrated to −20° C. and sectioned(20-30 μm sections on glass slides). Glass slides containing sectionedsamples were soaked in deionized water for at least 1 hour to removecryogel. Samples were permeabilized and blocked in blocking buffer for60 min., rinsed 2×15 min. with rinse buffer, and incubated at roomtemperature in incubation buffer for at least 60 min. Samples were thentreated with primary antibodies in incubation buffer at 4° C. (or atleast 4 hours at room temperature). Samples were then rinsed with washbuffer (2×15 min.) and incubation buffer (at least 60 minutes, roomtemperature). Samples were then treated with secondary antibodies and1:1000 DAPI (Sigma) in incubation buffer overnight at 4° C. (or at least2 hours at room temperature). Sectioned samples were mounted in aquapolymount solution (Polysciences, Inc.), a glass coverslip was placedover the top, stored overnight at 4° C., and sealed around the edgeswith fingernail sealant until imaging.

Image Processing: Confocal immunofluorescence images were collectedusing a Nikon MR confocal microscope. Images were processed using NISElements or ImageJ (Rasband 1997-2012; Schneider 2012). Some z-stackswere aligned using the “Align Current ND Document” (NIS Elements) or theStackReg plugin (ImageJ) before creating maximum projection images.

REFERENCES

-   1. D. Rice, S. Barone, Environ. Health Perspect. 108, 511 (June,    2000).-   2. L. Smirnova, H. T. Hogberg, M. Leist, T. Hartung, ALTEX-Altern.    Anim. Exp. 31, 129 (2014).-   3. P. Grandjean, P. J. Landrigan, Lancet Neurol. 13, 330 (March,    2014).-   4. L. L. Needham et al., Environ. Sci. Technol. 45, 1121 (February,    2011).-   5. P. Grandjean, P. J. Landrigan, Lancet 368, 2167 (December, 2006).-   6. D. C. Bellinger, Environ. Health Perspect. 120, 501 (April,    2012).-   7. Z. G. Hou et al., Stem Cell Res. Ther. 4, S12 (December, 2013).-   8. D. V. Hansen et al., Nat. Neurosci. 16, 1576 (November, 2013).-   9. J. H. Lui, D. V. Hansen, A. R. Kriegstein, Cell 146, 18 (July,    2011).-   10. P. Rakic, Nat. Rev. Neurosci. 10, 724 (October, 2009).-   11. I. Bystron, C. Blakemore, P. Rakic, Nat. Rev. Neurosci. 9, 110    (February, 2008).-   12. M. Marin-Padilla, Front. Neuroanat. 6, (September, 2012).-   13. M. Marin-Padilla, D. S. Knopman, J. Neuropathol. Exp. Neurol.    70, 1060 (December, 2011).-   14. J. M. James, Y.-s. Mukouyama, Semin. Cell Dev. Biol. 22, 1019    (2011).-   15. H. Stolp, A. Neuhaus, R. Sundramoorthi, Z. Molnar, Front.    Psychiatry 3, (2012).-   16. F. Ginhoux, S. Lim, G. Hoeffel, D. Low, T. Huber, Front. Cell.    Neurosci. 7, (April, 2013).-   17. T. Arnold, C. Betsholtz, Vascular Cell 5, 4 (2013).-   18. H. Kettenmann, U. K. Hanisch, M. Noda, A. Verkhratsky, Physiol.    Rev. 91, 461 (April, 2011).-   19. C. Verney, A. Monier, C. Fallet-Bianco, P. Gressens, J. Anat.    217, 436 (October, 2010).-   20. A. Monier et al., J. Neuropathol. Exp. Neurol. 66, 372 (May,    2007).-   21. A. Monier, P. Evrard, P. Gressens, C. Verney, J. Comp. Neurol.    499, 565 (December, 2006).-   22. J. A. Thomson et al., Science 282, 1145 (November, 1998).-   23. J. Y. Yu et al., Science 318, 1917 (December, 2007).-   24. K. Takahashi et al., Cell 131, 861 (November, 2007).-   25. S. C. Zhang, M. Wernig, I. D. Duncan, O. Brustle, J. A. Thomson,    Nat. Biotechnol. 19, 1129 (December, 2001).-   26. O. Brustle et al., Science 285, 754 (Jul. 30, 1999).-   27. M. A. Lancaster et al., Nature 501, 373 (September, 2013).-   28. J. Mariani et al., Proceedings of the National Academy of    Sciences 109, 12770 (Jul. 31, 2012, 2012).-   29. M. Eiraku et al., Cell Stem Cell 3, 519 (November, 2008).-   30. M. Ader, E. M. Tanaka, Curr. Opin. Cell Biol. 31, 23 (2014).-   31. I. Singec et al., Nat. Methods 3, 801 (October, 2006).-   32. B. D. Fairbanks et al., Adv. Mater. 21:5005-5010 (December,    2009).-   33. M. Marin-Padilla, Front. Neuroanat. 6, (2012 Sep. 13, 2012).-   34. P. Rakic, Cereb. Cortex 13, 541 (June, 2003).-   35. I. Bystron, P. Rakic, Z. Molnar, C. Blakemore, Nat Neurosci 9,    880 (2006).-   36. G. Meyer, J. P. Schaaps, L. Moreau, A. M. Goffinet, J Neurosci.    20, 1858 (March, 2000).-   37. N. Zecevic, A. Milosevic, S. Rakic, M. Marin-Padilla, The    Journal of Comparative Neurology 412, 241 (1999).-   38. M. H. Dominguez, A. E. Ayoub, P. Rakic, Cereb. Cortex 23, 2632    (November, 2013).-   39. B. J. Molyneaux, P. Arlotta, J. R. L. Menezes, J. D. Macklis,    Nat. Rev. Neurosci. 8, 427 (June, 2007).-   40. J. Struyf, S. Dobrin, D. Page, BMC Genomics 9, (November, 2008).-   41. T. R. Golub et al., Science 286, 531 (October, 1999).-   42. V. N. Vapnik, Statistical Learning Theory. (Wiley, New York,    1998), pp. 736.-   43. C. Cortes, V. Vapnik, Mach. Learn. 20, 273 (September, 1995).-   44. T. S. Furey et al., Bioinformatics 16, 906 (October, 2000).-   45. M. Moors et al., Environ. Health Perspect. 117, 1131 (July,    2009).-   46. N. C. Kleinstreuer et al., Nat. Biotechnol. 32, 583 (June,    2014).-   47. M. S. Wilson, J. R. Graham, A. J. Ball, Neurotoxicology 42, 33    (May, 2014).-   48. N. V. Balmer, M. Leist, Basic Clin. Pharmacol. Toxicol. 115, 59    (July, 2014).-   49. H. Olson et al., Regulatory Toxicology and Pharmacology 32, 56    (2000).-   50. S. Rakic, N. Zecevic, Cereb. Cortex 13, 1072 (October, 2003).-   51. T. Kadoshima et al., Proc. Natl. Acad. Sci. U.S.A 110, 20284    (December, 2013).-   52. G. K. Chen et al., Nat. Methods 8, 424 (May, 2011).-   53. X. J. Li et al., Development 136, 4055 (December, 2009).-   54. S. M. Chambers et al., Nat. Biotechnol. 27, 275 (March, 2009).-   55. H. Nagase, G. B. Fields, Biopolymers 40, 399 (1996).-   56. M. D. Pierschbacher, E. Ruoslahti, Nature 309, 30 (1984).-   57. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Genome Biol    10, R25 (2009).-   58. B. Li, C. N. Dewey, BMC Bioinformatics 12, 323 (2011).

We claim:
 1. A method of making a composition comprising humanmicroglial cells, the method comprising culturing human pluripotent stemcell-derived CD45⁺/CD11b⁺/CD14⁺ primitive macrophages for at least fivedays in a chemically defined hydrogel-based three-dimensional tissueconstruct comprising stratified layers of human neurons and glia,thereby producing a composition comprising human microglial cellsexpressing CD68, CD11b, Triggering Receptor Expressed on Myeloid Cells 2(TREM2), and Ionized calcium-binding adapter molecule 1 (Iba1).
 2. Themethod of claim 1, wherein the primitive macrophages are obtained byculturing human myeloid progenitors in the presence of a culture mediumcomprising insulin and a hematopoietic cytokine, whereby the culturedmyeloid progenitors differentiate into a cell population comprising atleast 80% CD45⁺/CD11b⁺/CD14⁺ primitive macrophages, wherein the humanmyeloid progenitors are obtained by (a) culturing human pluripotent stemcells under normoxic conditions for about 24 hours, wherein thepluripotent stem cells are cultured on a substrate comprisingrecombinant human Tenascin-C and in the presence of a culture mediumconsisting essentially of L-ascorbic acid-2-phosphate magnesium, sodiumselenium, transferrin, insulin, NaHCO₃, fibroblast growth factor 2(FGF2), transforming growth factor beta 1 (TGF(α1), and a Rho kinase(ROCK) inhibitor, whereby the cultured pluripotent stem cellsdifferentiate into hematopoietic precursor cells (HPCs); and (b)culturing the HPCs obtained in (a) for about 3 to about 5 days in aculture medium comprising FGF2, a vascular endothelium growth factor(VEGF), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6(IL-6), and interleukin-3 (IL-3), wherein the hematopoietic progenitorcells differentiate into myeloid progenitors.
 3. The method of claim 1,wherein, prior to the contacting step, the human primitive macrophagesare cultured for about 5 days in a culture medium consisting essentiallyof Iscove's Modified Dulbecco's Media (IMDM), interleukin-1-beta(IL-1α), serum, and a hematopoietic growth factor.
 4. The method ofclaim 1, wherein the hematopoietic growth factor is macrophagecolony-stimulating factor (M-CSF).
 5. A method of producing humanmicroglial cells, wherein the method comprises culturing humanpluripotent stem cell-derived CD45⁺/CD11b⁺/CD14⁺ primitive macrophagesfor at least five days in a chemically defined culture medium comprisingIscove's Modified Dulbecco's Media (IMDM), interleukin-1-beta (IL-1α),serum, and a hematopoietic growth factor, whereby a cell populationcomprising human microglial cells expressing CD68, CD11b, TriggeringReceptor Expressed on Myeloid Cells 2 (TREM2), and Ionizedcalcium-binding adapter molecule 1 (Iba1) is produced.
 6. The method ofclaim 5, wherein the chemically defined culture medium consistsessentially of IMDM, IL-1α, serum, and macrophage colony-stimulatingfactor (M-CSF).
 7. The method of claim 5, wherein the primitivemacrophages are obtained by culturing human myeloid progenitors in thepresence of a culture medium comprising insulin and a hematopoieticcytokine, whereby the cultured myeloid progenitors differentiate into acell population comprising at least 80% CD45⁺/CD11b⁺/CD14⁺ primitivemacrophages, wherein the human myeloid progenitors are obtained by (a)culturing human pluripotent stem cells under normoxic conditions forabout 24 hours, wherein the pluripotent stem cells are cultured on asubstrate comprising recombinant human Tenascin-C and in the presence ofa culture medium consisting essentially of L-ascorbic acid-2-phosphatemagnesium, sodium selenium, transferrin, insulin, NaHCO₃, fibroblastgrowth factor 2 (FGF2), transforming growth factor beta 1 (TGFα1), and aRho kinase (ROCK) inhibitor, whereby the cultured pluripotent stem cellsdifferentiate into hematopoietic precursor cells (HPCs); and (b)culturing the HPCs obtained in (a) for about 3 to about 5 days in aculture medium comprising FGF2, a vascular endothelium growth factor(VEGF), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6(IL-6), and interleukin-3 (IL-3), wherein the hematopoietic progenitorcells differentiate into myeloid progenitors.